Dynamics of synaptic damage in severe traumatic brain injury revealed by cerebrospinal fluid SNAP-25 and VILIP-1.

BACKGROUND: Biomarkers of neuronal, glial cells and inflammation in traumatic brain injury (TBI) are available but they do not specifically reflect the damage to synapses, which represent the bulk volume of the brain. Experimental models have demonstrated extensive involvement of synapses in acute TBI, but biomarkers of synaptic damage in human patients have not been explored. METHODS: Single-molecule array assays were used to measure synaptosomal-associated protein-25 (SNAP-25) and visinin-like protein 1 (VILIP-1) (along with neurofilament light chain (NFL), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillar acidic protein (GFAP), interleukin-6 (IL-6) and interleukin-8 (IL-8)) in ventricular cerebrospinal fluid (CSF) samples longitudinally acquired during the intensive care unit (ICU) stay of 42 patients with severe TBI or 22 uninjured controls. RESULTS: CSF levels of SNAP-25 and VILIP-1 are strongly elevated early after severe TBI and decline in the first few days. SNAP-25 and VILIP-1 correlate with inflammatory markers at two distinct timepoints (around D1 and then again at D5) in follow-up. SNAP-25 and VILIP-1 on the day-of-injury have better sensitivity and specificity for unfavourable outcome at 6 months than NFL, UCH-L1 or GFAP. Later elevation of SNAP-25 was associated with poorer outcome. CONCLUSION: Synaptic damage markers are acutely elevated in severe TBI and predict long-term outcomes, as well as, or better than, markers of neuroaxonal injury. Synaptic damage correlates with initial injury and with a later phase of secondary inflammatory injury.

The tissue-type plasminogen activator-plasminogen activator inhibitor 1 complex promotes neurovascular injury in brain trauma: evidence from mice and humans.

The neurovascular unit provides a dynamic interface between the circulation and central nervous system. Disruption of neurovascular integrity occurs in numerous brain pathologies including neurotrauma and ischaemic stroke. Tissue plasminogen activator is a serine protease that converts plasminogen to plasmin, a protease that dissolves blood clots. Besides its role in fibrinolysis, tissue plasminogen activator is abundantly expressed in the brain where it mediates extracellular proteolysis. However, proteolytically active tissue plasminogen activator also promotes neurovascular disruption after ischaemic stroke; the molecular mechanisms of this process are still unclear. Tissue plasminogen activator is naturally inhibited by serine protease inhibitors (serpins): plasminogen activator inhibitor-1, neuroserpin or protease nexin-1 that results in the formation of serpin:protease complexes. Proteases and serpin:protease complexes are cleared through high-affinity binding to low-density lipoprotein receptors, but their binding to these receptors can also transmit extracellular signals across the plasma membrane. The matrix metalloproteinases are the second major proteolytic system in the mammalian brain, and like tissue plasminogen activators are pivotal to neurological function but can also degrade structures of the neurovascular unit after injury. Herein, we show that tissue plasminogen activator potentiates neurovascular damage in a dose-dependent manner in a mouse model of neurotrauma. Surprisingly, inhibition of activity following administration of plasminogen activator inhibitor-1 significantly increased cerebrovascular permeability. This led to our finding that formation of complexes between tissue plasminogen activator and plasminogen activator inhibitor-1 in the brain parenchyma facilitates post-traumatic cerebrovascular damage. We demonstrate that following trauma, the complex binds to low-density lipoprotein receptors, triggering the induction of matrix metalloproteinase-3. Accordingly, pharmacological inhibition of matrix metalloproteinase-3 attenuates neurovascular permeability and improves neurological function in injured mice. Our results are clinically relevant, because concentrations of tissue plasminogen activator: plasminogen activator inhibitor-1 complex and matrix metalloproteinase-3 are significantly elevated in cerebrospinal fluid of trauma patients and correlate with neurological outcome. In a separate study, we found that matrix metalloproteinase-3 and albumin, a marker of cerebrovascular damage, were significantly increased in brain tissue of patients with neurotrauma. Perturbation of neurovascular homeostasis causing oedema, inflammation and cell death is an important cause of acute and long-term neurological dysfunction after trauma. A role for the tissue plasminogen activator-matrix metalloproteinase axis in promoting neurovascular disruption after neurotrauma has not been described thus far. Targeting tissue plasminogen activator: plasminogen activator inhibitor-1 complex signalling or downstream matrix metalloproteinase-3 induction may provide viable therapeutic strategies to reduce cerebrovascular permeability after neurotrauma.

Rod microglia: a morphological definition.

Brain microglial morphology relates to function, with ramified microglia surveying the micro-environment and amoeboid microglia engulfing debris. One subgroup of microglia, rod microglia, have been observed in a number of pathological conditions, however neither a function nor specific morphology has been defined. Historically, rod microglia have been described intermittently as cells with a sausage-shaped soma and long, thin processes, which align adjacent to neurons. More recently, our group has described rod microglia aligning end-to-end with one another to form trains adjacent to neuronal processes. Confusion in the literature regarding rod microglia arises from some reports referring to the sausage-shaped cell body, while ignoring the spatial distribution of processes. Here, we systematically define the morphological characteristics of rod microglia that form after diffuse brain injury in the rat, which differ morphologically from the spurious rod microglia found in uninjured sham. Rod microglia in the diffuse-injured rat brain show a ratio of 1.79 ± 0.03 cell length:cell width at day 1 post-injury, which increases to 3.35 ± 0.05 at day 7, compared to sham (1.17 ± 0.02). The soma length:width differs only at day 7 post-injury (2.92 ± 0.07 length:width), compared to sham (2.49 ± 0.05). Further analysis indicated that rod microglia may not elongate in cell length but rather narrow in cell width, and retract planar (side) processes. These morphological characteristics serve as a tool for distinguishing rod microglia from other morphologies. The function of rod microglia remains enigmatic; based on morphology we propose origins and functions for rod microglia after acute neurological insult, which may provide biomarkers or therapeutic targets.

Nuclear trafficking of Pten after brain injury leads to neuron survival not death.

There is controversy whether accumulation of the tumor suppressor PTEN protein in the cell nucleus under stress conditions such as trauma and stroke causes cell death. A number of in vitro studies have reported enhanced apoptosis in neurons possessing nuclear PTEN, with the interpretation that its nuclear phosphatase activity leads to reduction of the survival protein phospho-Akt. However, there have been no in vivo studies to show that nuclear PTEN in neurons under stress is detrimental. Using a mouse model of injury, we demonstrate here that brain trauma altered the nucleo-cytoplasmic distribution of Pten, resulting in increased nuclear Pten but only in surviving neurons near the lesion. This event was driven by Ndfip1, an adaptor and activator of protein ubiquitination by Nedd4 E3 ligases. Neurons next to the lesion with nuclear PTEN were invariably negative for TUNEL, a marker for cell death. These neurons also showed increased Ndfip1 which we previously showed to be associated with neuron survival. Biochemical assays revealed that overall levels of Pten in the affected cortex were unchanged after trauma, suggesting that Pten abundance globally had not increased but rather Pten subcellular location in affected neurons had changed. Following experimental injury, the number of neurons with nuclear Pten was reduced in heterozygous mice (Ndfip1(+/-)) although lesion volumes were increased. We conclude that nuclear trafficking of Pten following injury leads to neuron survival not death.

Ablation of D1 dopamine receptor-expressing cells generates mice with seizures, dystonia, hyperactivity, and impaired oral behavior.

Huntington's disease is characterized by death of striatal projection neurons. We used a Cre/Lox transgenic approach to generate an animal model in which D1 dopamine receptor (Drd1a)+ cells are progressively ablated in the postnatal brain. Striatal Drd1a, substance P, and dynorphin expression is progressively lost, whereas D2 dopamine receptor (Drd2) and enkephalin expression is up-regulated. Magnetic resonance spectroscopic analysis demonstrated early elevation of the striatal choline/creatine ratio, a finding associated with extensive reactive striatal astrogliosis. Sequential MRI demonstrated a progressive reduction in striatal volume and secondary ventricular enlargement confirmed to be due to loss of striatal cells. Mutant mice had normal gait and rotarod performance but displayed hindlimb dystonia, locomotor hyperactivity, and handling-induced electrographically verified spontaneous seizures. Ethological assessment identified an increase in rearing and impairments in the oral behaviors of sifting and chewing. In line with the limbic seizure profile, cell loss, astrogliosis, microgliosis, and down-regulated dynorphin expression were seen in the hippocampal dentate gyrus. This study specifically implicates Drd1a+ cell loss with tail suspension hindlimb dystonia, hyperactivity, and abnormal oral function. The latter may relate to the speech and swallowing disturbances and the classic sign of tongue-protrusion motor impersistence observed in Huntington's disease. In addition, the findings of this study support the notion that Drd1a and Drd2 are segregated on striatal projection neurons.